Standards in Genomic Sciences (2011) 5:287-297 DOI:10.4056/sigs.2205004

The Genomic Standards Consortium

Complete genome sequence of Hirschia baltica type strain (IFAM 1418T) Olga Chertkov1,2, Pamela J.B. Brown3, David T. Kysela3, Miguel A. DE Pedro4, Susan Lucas1, Alex Copeland1, Alla Lapidus1, Tijana Glavina Del Rio1, Hope Tice1, David Bruce1, Lynne Goodwin1,2, Sam Pitluck1, John C. Detter1,2, Cliff Han1,2, Frank Larimer2, Yun-juan Chang1,5, Cynthia D. Jeffries1,5, Miriam Land1,5, Loren Hauser1,5, Nikos C. Kyrpides1, Natalia Ivanova1, Galina Ovchinnikova1, Brian J. Tindall6, Markus Göker6, Hans-Peter Klenk6*, Yves V. Brun3* 1 DOE Joint Genome Institute, Walnut Creek, California, USA 2 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 3 Indiana University, Bloomington, Indiana, USA 4 Universidad Autonoma de Madrid, Campus de Cantoblanco, Madrid, Spain 5 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 6 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig,

Germany

*Corresponding author: hpk@dsmz.de, ybrun@indiana.edu

Keywords: aerobic, chemoheterotrophic, mesophile, Gram-negative, motile, budding, stalk-forming, Hyphomonadaceae, Alphaproteobacteria, CSP 2008

The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacte-ria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. bal-tica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacte-rium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 pro-tein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008.

Introduction Strain IFAM 1418T (= ATCC 49814 = DSM 5838 = KCTC 12471) is the type strain of the species Hir-schia baltica, which is the type species of the ge-nus Hirschia. The genus name Hirschia Baker 1896 has been applied to a member of the Aste-raceae, although the type and only species, Hir-schia anthemidifolia has also been placed in the genus Iphiona [1]. The genus Hirschia was named in honor of Peter Hirsch, a German microbiolo-gist and expert on budding and hyphal bacteria. The species epithet refers to the Neo-Latin balti-ca, pertaining to the Baltic Sea [2]. IFAM 1418T was isolated in 1982 along with two additional stalk-forming isolates belonging to the same spe-cies, IFAM 1408 and IFAM 1415, from a sample of surface water taken from a boat landing in the Kiel Fjord [2]. H. baltica reproduces by budding from the tip of a stalk and has a dimorphic life-cycle similar to that described in bacteria belonging to the

closely related genus Hyphomonas [2]. Newborn swarmer cells are motile by means of a polar fla-gellum. Swarmer cells differentiate into stalked sessile cells by ejecting the flagellum and synthe-sizing a stalk at the same pole. The sessile cells reproduce by budding such that motile daughter cells are formed at and released from the tip of the stalk. Here we present a summary classifica-tion and a set of features for H. baltica IFAM 1418T, together with the description of the com-plete genomic sequencing and annotation.

Classification and features A representative genomic 16S rRNA sequence of H. baltica was compared using NCBI BLAST [3] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [4] and the relative frequen-cies of taxa and keywords (reduced to their stem

Hirschia baltica type strain (IFAM 1418T)

288 Standards in Genomic Sciences

[5]) were determined, weighted by BLAST scores. The most frequently occurring genera were Hy-phomonas (37.8%), Maricaulis (29.5%), Hirschia (14.3%), Caulobacter (7.4%) and Woodsholea (3.6%) (74 hits in total). Regarding the four hits to sequences from members of the Hirschia species, the average identity within HSPs was 99.3%, whe-reas the average coverage by HSPs was 98.8%. Among all other species, the one yielding the high-est score was Hyphomonas johnsonii (NR_024938), which corresponded to an identity of 93.1% and an HSP coverage of 59.6%. (Note that the Green-genes database uses the INSDC (= EMBL /NCBI/DDBJ) annotation, which is not an authori-tative source for nomenclature or classification.) The highest-scoring environmental sequence was FR684125 ('effect ocean acidification upon micro-bial prokaryotes marine biome fjord coastal water

clone 16 07 04A09'), which showed an identity of 89.4% and an HSP coverage of 99.0%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'marin' (2.2%), 'microbi' (2.1%), 'water' (2.0%), 'biofilm' (1.9%) and 'sea' (1.6%) (176 hits in to-tal). Environmental samples which yielded hits of a higher score than the highest scoring species were not found. These keywords reflect very well some the ecological and physiological properties reported for strain IFAM 1418T in the original de-scription [2]. Figure 1 shows the phylogenetic neighborhood of H. baltica in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by one nucleotide from the pre-viously published 16S rRNA sequence (AJ421782), which contains one ambiguous base call.

Figure 1. Phylogenetic tree highlighting the position of H. baltica relative to the type strains of the other species with-in the family Hyphomonadaceae. The tree was inferred from 1,332 aligned characters [6,7] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [8] and rooted with the neighboring family Parvularculaceae. The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 450 ML bootstrap replicates [9] (left) and from 1,000 maximum parsimony (MP) bootstrap replicates [10] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [11] are labeled with one asterisk, those also listed as 'Complete and Published' (as well as the target genome) with two asterisks [12,13].

Chertkov et al.

http://standardsingenomics.org 289

Figure 2. Scanning Electron micrograph of H. baltica IFAM 1418T

Cells of H. baltica strain IFAM 1418T are rod-shaped, elliptical or ovoid with 0.5 – 1.0 by 0.5 – 6.0 µm in size (without hyphae which have a di-ameter of about 0.2 µm) (Figure 2) [2]. IFAM 1418T cells stain Gram-negative, are motile and strictly aerobic [2]. 1 to 2 hyphae are located po-larly and flagellation is monotrichous polar [2]. Cells grow best in artificial sea water with a broad range of NaCl concentrations (Table 1). The strain grows on a broad spectrum of organic compounds as carbon source such as amino acids, organic ac-ids and sugars (Table 1), but not on C1 compounds [2]. In contrast to its phylogenetic neighbors H. baltica strains do not store PHB [2]. Colonies of strain IFAM 1418T show a yellow pigmentation whereas strain IFAM 1408 colonies are white [2].

Chemotaxonomy Cell walls of H. baltica contain meso-diamonopimelic acid [2]. The main quinone com-ponent is Q10 [2,24]. In the original publications by Schlesner et al. [2] and Sittig and Hirsch [24] the fatty acids were separated into non-hydroxy fatty acids and hydroxy-fatty acids. Main cellular non-hydroxy fatty acids are C18:1 Δ11 (48.8%), C16:0 (22.1%), C18:2 Δ5,11 (9.5%), C16:1 Δ5 (4.6%), and C14:1 Δ7 (3.2%). Among the hydroxylated fatty acids IFAM 1418T 3-OH fatty acids with C14:1 3-OH (79%) and C12:0 3-OH (15.8%) predominate, which are use-ful for the discrimination for H. baltica from strains of Hyphomicrobium and Hyphomonas (Fig-ure 1). Kang and Lee [25] were unable to confirm the presence of these hydoxylated fatty acids in H. baltica. There are two reports [2,24] on the lipid composition of H. baltica and one on the lipids of H. maritima [25]. While all confirm the presence of

a single phospholipid, phosphatidylglycerol in members of this genus, it is unclear whether these studies were limited to the detection of only phospholipids. There is evidence that H. baltica contains five major lipids, of which one is phosphatidlyglycerol (BJ Tindall, unpublished). The remaining four lipids are not phosphate posi-tive, but their Rf values and staining behavior indi-cate similarities to a number of the unusual lipids reported from Hyphomonas jannaschiana [26,27].

Genome sequencing and annotation Genome project history This organism was selected for sequencing as a part of the DOE Joint Genome Institute Program CSP 2008. The genome project is deposited in the Genomes On Line Database [11] and the complete genome sequence is deposited in GenBank. Se-quencing, finishing and annotation were per-formed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Strain history The history of strain IFAM 1418T starts with H. Schlesner who independently deposited the strain in two culture collections, ATCC and DSMZ (in 1989).

Growth conditions and DNA isolation The culture of strain IFAM 1418T used to prepare genomic DNA (gDNA) for sequencing was ob-tained directly from the ATCC. ATCC 49814 was grown in Hirschia medium (ATCC medium 1883) at 30°C with shaking.

http://standardsingenomics.org/�

Hirschia baltica type strain (IFAM 1418T)

290 Standards in Genomic Sciences

Table 1. Classification and general features of H. baltica according to the MIGS recommendations [14]. MIGS ID Property Term Evidence code

Current classification

Domain Bacteria TAS [15] Phylum Proteobacteria TAS [16] Class Alphaproteobacteria TAS [17,18] Order Caulobacterales TAS [19,20] Family Hyphomonadaceae TAS [21] Genus Hirschia TAS [2] Species Hirschia baltica TAS [2] Type strain IFAM 1418 TAS [2]

Gram stain negative TAS [2] Cell shape rod-shaped TAS [2] Motility motile TAS [2] Sporulation none TAS [2] Temperature range mesophile TAS [2] Optimum temperature 22-28°C TAS [2] Salinity artificial sea water, 0.5 – 8.6% NaCl TAS [2] MIGS-22 Oxygen requirement aerobe TAS [2] Carbon source various amino acids, organic acids and sugars TAS [2] Energy metabolism chemoheterotroph TAS [2] MIGS-6 Habitat brackish water TAS [2] MIGS-15 Biotic relationship free living TAS [2] MIGS-14 Pathogenicity none NAS Biosafety level 1 TAS [22] Isolation surface water samples TAS [2] MIGS-4 Geographic location Baltic Sea, Kiel Fjord TAS [2] MIGS-5 Sample collection time October 1982 NAS MIGS-4.1 Latitude approximately 54.5 NAS MIGS-4.2 Longitude approximately 10.2 NAS MIGS-4.3 Depth 5 cm TAS [2] MIGS-4.4 Altitude sea level NAS

Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [23].

Genome sequencing and assembly The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [28]. Pyrosequencing reads were assembled using the Newbler assemb-ler (Roche). The initial Newbler assembly consist-ing of nine contigs in two scaffolds was converted into a phrap [29] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (50 Mb) were assembled with Velvet [30] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together

with the 454 data. The 454 draft assembly was based on 128.0 Mb 454 draft data and all of the 454 paired end data. Newbler parameters were -consed -a 50 - l350 -g -m -ml 20. The Phred/Phrap/Consed software package [29] was used for sequence as-sembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Perfor-mance Software, LLC). Possible mis-assemblies were corrected with gapResolution [28], Dupfi-nisher [31], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by

Chertkov et al.

http://standardsingenomics.org 291

Bubble PCR primer walks (J.-F. Chang, unpub-lished). A total of 55 additional reactions were ne-cessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consen-sus quality using a software Polisher developed at

JGI [28,32]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 150.6 × coverage of the ge-nome. The final assembly contained 333,683 pyro-sequence and 12,860,398 Illumina reads.

Table 2. Genome sequencing project information MIGS ID Property Term MIGS-31 Finishing quality Finished

MIGS-28 Libraries used Three genomic libraries: one 454 pyrosequence standard li-brary, one 454 PE library (24 kb insert size), one Illumina library

MIGS-29 Sequencing platforms 454 GS FLX Titanium, Illumina GAii MIGS-31.2 Sequencing coverage 116.6 × Illumina; 34.0 x pyrosequence MIGS-30 Assemblers Newbler version 2.0.00.20, Velvet, phrap MIGS-32 Gene calling method Critica complemented with the output of Glimmer

INSDC ID CP001678 (chromosome)

CP001679 (plasmid pHbal01) GenBank Date of Release July 19, 2009 GOLD ID Gc01064 NCBI project ID 33191 Database: IMG 644736375 MIGS-13 Source material identifier ATCC 49814 Project relevance Bioremediation, Biotechnology

Genome annotation Genes were identified using Prodigal [33] as part of the Oak Ridge National Laboratory genome an-notation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [34]. The predicted CDSs were translated and used to search the National Center for Biotechnology In-formation (NCBI) non-redundant database, Uni-Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. These data sources were com-bined to assert a product description for each predicted protein. Non-coding genes and miscel-laneous features were predicted using tRNAscan-SE [35], RNAMMer [36], Rfam [37], TMHMM [38], and signalP [39].

Genome properties The genome consists of a 3,455,622 bp long chro-mosome with a 45% G+C content and a 84,492 bp long plasmid with a 44% G+C content (Table 3 and Figure 3). Of the 3,266 genes predicted, 3,222 were protein-coding genes, and 44 RNAs; 35 pseudo-genes were also identified. The majority of the pro-tein-coding genes (71.6%) were assigned a puta-tive function while the remaining ones were anno-tated as hypothetical proteins. The distribution of

genes into COGs functional categories is presented in Table 4.

Notable features of the H. baltica genome sequence H. baltica genome is enriched in TonB-dependent receptors TonB-dependent receptors are outer membrane proteins that function in the energy-dependent up-take of macromolecules, including siderophores and vitamins, that are too large to be taken up by passive diffusion. The genome sequences of C. cres-centus and H. neptunium contain large numbers (63 and 43, respectively) of TonB dependent receptors [40,41]. One possibility is that the presence of many TonB-dependent receptors is a common fea-ture among bacteria belonging to the order Caulo-bacterales. Indeed, the annotation of the H. baltica genome sequence suggests that there are 46 TonB-dependent receptors. The presence of many TonB-dependent receptors may be part of the adapta-tions that have allowed the Caulobacterales to not only inhabit, but also thrive in low-nutrient envi-ronments.

http://standardsingenomics.org/�

Hirschia baltica type strain (IFAM 1418T)

292 Standards in Genomic Sciences

Table 3. Genome Statistics Attribute Value % of Total Genome size (bp) 3,540,114 100.00% DNA coding region (bp) 3,172,944 89.63% DNA G+C content (bp) 1,599,770 45.19% Number of replicons 2 Extrachromosomal elements 1 Total genes 3,266 100.00% RNA genes 44 1.35% rRNA operons 2 Protein-coding genes 3,222 98.65% Pseudo genes 35 1.07% Genes with function prediction 2,337 71.65% Genes in paralog clusters 223 6.83% Genes assigned to COGs 2,443 74.80% Genes assigned Pfam domains 2,551 78.11% Genes with signal peptides 797 24.40% Genes with transmembrane helices 759 23.24% CRISPR repeats not reported

H. baltica genome contains known regulators of the cell cycle A recent bioinformatic analysis of genes controlling the dimorphic cell cycle within the Alphaproteobac-teria suggests the circuitry for cell-cycle control is largely conserved [42]. The conservation of 14 key proteins that function in the regulation of the cell cycle in C. crescentus was addressed among the Al-phaproteobacteria with sequenced genomes, includ-ing three bacteria belonging to the Caulobacterales. All of the regulatory proteins were conserved among the genomes of the Caulobacterales with one notable exception: DivJ, a histidine kinase, is absent in the H. neptunium genome. Expanding the analysis to include 8 additional bacte-ria belonging to the Caulobacterales, including H. baltica, we find that all 14 regulatory proteins are conserved with the exception of DivJ, which is ab-sent only from the H. neptunium and H. baltica ge-nomes. The fact that most developmental regulators are conserved in the budding bacteria H. neptunium and H. baltica as well as the non-budding bacteria belonging to the Caulobacterales suggests that regu-lation of the cell cycle is evolved prior to the separa-tion of the budding and non-budding bacteria in the Caulobacterales. The finding that DivJ is absent from H. neptunium and H. baltica but present in the close-ly related non-budding Maricaulis maris and Oceani-caulis alexandrii (Figure 1) is an intriguing observa-tion, although the significance of this finding remains unknown.

H. baltica genome contains genes for hold-fast synthesis and attachment Bacteria belonging to the order Caulobacterales are known for the ability to produce a polar polysaccha-ride, termed holdfast, which mediates strong adhe-sion to surfaces (For review see [43]). Notably, extracellular polysaccharides from some of the stalked bacteria sequester metals [44,45], a feature that could be used to remediate environments af-fected by metal toxicity. The genes required for the synthesis [46,47] and anchoring [48] of the holdfast have been identified and characterized in C. crescen-tus. The holdfast synthesis and anchor genes are largely absent from the genome of H. neptunium, which does not produce a polar holdfast (Figure 4 and [41]). The genome sequence of H. baltica re-vealed that the genes predicted to be involved in po-lar holdfast synthesis are present (Figure 4). Fur-thermore, a holdfast on H. baltica cells was readily detected using a fluorescent wheat germ agglutinin lectin using the procedure detailed in [44] (Figure 4). The holdfast of H. baltica is found at the cell pole opposite the stalk of the mother cell and is responsi-ble for the formation of star-shaped cell aggregates known as rosettes in cell culture (Figure 4). This finding, coupled with the observation that other spe-cies of Hyphomonas produce detectable holdfasts [49], suggests the ability to synthesize holdfast is a conserved feature among the Hyphomonadaceae family and the loss of the holdfast synthesis and anchoring genes in H. neptunium was a recent evolu-tionary event.

Chertkov et al.

http://standardsingenomics.org 293

Figure 3. Graphical circular map of the chromosome (plasmid not shown). From outside to the center: Genes on for-ward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

http://standardsingenomics.org/�

Hirschia baltica type strain (IFAM 1418T)

294 Standards in Genomic Sciences

Table 4. Number of genes associated with the general COG functional categories Code value %age Description

J 158 5.9 Translation, ribosomal structure and biogenesis A 0 0.0 RNA processing and modification K 167 6.2 Transcription L 106 3.9 Replication, recombination and repair B 1 0.0 Chromatin structure and dynamics D 28 1.0 Cell cycle control, cell division, chromosome partitioning Y 0 0.0 Nuclear structure V 34 1.3 Defense mechanisms T 140 5.2 Signal transduction mechanisms M 196 7.3 Cell wall/membrane biogenesis N 70 2.6 Cell motility Z 0 0.0 Cytoskeleton W 0 0.0 Extracellular structures U 92 3.4 Intracellular trafficking and secretion, and vesicular transport O 129 4.8 Posttranslational modification, protein turnover, chaperones C 152 5.7 Energy production and conversion G 141 5.2 Carbohydrate transport and metabolism E 204 7.6 Amino acid transport and metabolism F 55 2.1 Nucleotide transport and metabolism H 119 4.4 Coenzyme transport and metabolism I 133 5.0 Lipid transport and metabolism P 152 5.7 Inorganic ion transport and metabolism Q 82 3.1 Secondary metabolites biosynthesis, transport and catabolism R 287 10.7 General function prediction only S 243 9.0 Function unknown - 823 25.2 Not in COGs

Figure 4. Holdfast anchor (hfa) and synthesis (hfs) gene cluster con-servation is depicted among C. crescentus, H. baltica and H. neptu-nium. Presence of holdfast genes is correlated to the ability to detect polar holdfast polysaccharide using fluorescent wheat germ aggluti-nin lectin. The H. neptunium genome contains only hfsAB and fails to make a polar polysaccharide. In contrast, the H. baltica genome contains all essential hfs and hfa genes and produces a holdfast.

Chertkov et al.

http://standardsingenomics.org 295

Concluding Remarks The completion of the H. baltica genome sequence is expected to provide a valuable resource for un-derstanding the biology of stalked budding bacteria with a dimorphic life cycle. Further comparative genomic analyses (especially as more bacteria be-longing to the Caulobacterales are sequenced) are likely to provide insights regarding the evolution,

mechanism, and advantages underlying processes such as budding, and stalk synthesis. Understand-ing stalk biosynthesis is of particular interest as the ability of these structures to transport nutrients [50,51] could likely be exploited to facilitate the uptake of toxic compounds from contaminated wa-ter sources.

Acknowledgements This work was supported by a Department of Energy Joint Genome Institute (DOE-JGI) 2008 Community Se-quencing Project (787681) award to Y.V.B. The work conducted by the U.S. DOE-JGI was supported by the Office of Science of the U.S. Department of Energy un-der Contract No. DE-AC02-05CH11231. This work was also supported by National Institutes of Health (GM051986 and GM077648) and National Science Foundation (MCB0731950) awards to Y.V.B. P.J.B.B.

and D.T.K. were supported by National Institutes of Health National Research Service Awards AI072992 and GM083581, respectively. M.A.P. was supported by Ministry of Education and Science, Spain (MEC, BFU2006-04574) and Fundación Ramón Areces. Part of this work was also supported by a grant from the Indi-ana University META-Cyt program funded in part by a major endowment from the Lilly Foundation.

References 1. Anderberg A. The genus Iphiona (Compositae-

Inuleae). Nord J Bot 1985; 5:169-194. doi:10.1111/j.1756-1051.1985.tb02086.x

2. Schlesner H, Bartels C, Sittig M, Dorsch M, Stack-ebrandt E. Taxonomic and phylogenetic studies on a new taxon of budding, hyphal Proteobacte-ria, Hirschia baltica gen. nov., sp. nov. Int J Syst Bacteriol 1990; 40:443-451. PubMed doi:10.1099/00207713-40-4-443

3. Altschul SF, Gish W, Miller W, Myers EW, Lip-man DJ. Bascic local alignment search tool. J Mol Biol 1990; 215:403-410. PubMed

4. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compat-ible with ARB. Appl Environ Microbiol 2006; 72:5069-5072. PubMed doi:10.1128/AEM.03006-05

5. Porter MF. An algorithm for suffix stripping. Pro-gram: electronic library and information systems 1980; 14:130-137.

6. Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformat-ics 2002; 18:452-464. PubMed doi:10.1093/bioinformatics/18.3.452

7. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540-552. PubMed

8. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758-771. PubMed doi:10.1080/10635150802429642

9. Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184-200. doi:10.1007/978-3-642-02008-7_13

10. Swofford DL. PAUP*: Phylogenetic Analysis Us-ing Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.

11. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: Status of genomic and metage-nomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346-D354. PubMed doi:10.1093/nar/gkp848

12. Badger JH, Hoover TR, Brun YV, Weiner RM, Laub MT, Alexandre G, Mrázek J, Ren Q, Paulsen IT, Nelson KE, et al. Comparative genomic evi-dence for a close relationship between the di-morphic prosthecate bacteria Hyphomonas nep-tunium and Caulobacter crescentus. J Bacteriol 2006; 188:6841-6850. PubMed doi:10.1128/JB.00111-06

13. Oh HM, Kang I, Vergin KL, Kang D, Rhee KH, Giovannoni SJ, Cho JC. Complete Genome Se-quence of Strain HTCC2503T of Parvularcula bermudensis, the Type Species of the Order 'Parvularculales' in the Class Alphaproteobacte-

http://standardsingenomics.org/�http://dx.doi.org/10.1111/j.1756-1051.1985.tb02086.x�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2275859&dopt=Abstract�http://dx.doi.org/10.1099/00207713-40-4-443�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2231712&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16820507&dopt=Abstract�http://dx.doi.org/10.1128/AEM.03006-05�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11934745&dopt=Abstract�http://dx.doi.org/10.1093/bioinformatics/18.3.452�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10742046&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10742046&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10742046&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=18853362&dopt=Abstract�http://dx.doi.org/10.1080/10635150802429642�http://dx.doi.org/10.1007/978-3-642-02008-7_13�http://dx.doi.org/10.1007/978-3-642-02008-7_13�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19914934&dopt=Abstract�http://dx.doi.org/10.1093/nar/gkp848�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16980487&dopt=Abstract�http://dx.doi.org/10.1128/JB.00111-06�

Hirschia baltica type strain (IFAM 1418T)

296 Standards in Genomic Sciences

ria. J Bacteriol 2011; 193:305-306. PubMed doi:10.1128/JB.01205-10

14. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, An-giuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541-547. PubMed doi:10.1038/nbt1360

15. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms. Proposal for the do-mains Archaea and Bacteria. Proc Natl Acad Sci USA 1990; 87:4576-4579. PubMed doi:10.1073/pnas.87.12.4576

16. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Pro-teobacteria phyl. nov. In: DJ Brenner, NR Krieg, JT Staley, GM Garrity (eds), Bergey's Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria), part B (The Gammaproteo-bacteria), Springer, New York, 2005, p. 1.

17. Validation List No. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2006; 56:1-6. PubMed doi:10.1099/ijs.0.64188-0

18. Garrity GM, Bell JA, Lilburn T. Class I. Alphapro-teobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 1.

19. Henrici AT, Johnson DE. Studies of Freshwater Bacteria: II. Stalked bacteria, a New Order of Schizomycetes. J Bacteriol 1935; 30:61-93.

20. Skerman VDB, Gowan V, Sneath PHA, eds. Ap-proved Lists of Bacterial Names. Int J Syst Bacte-riol 1980; 30:225-420. doi:10.1099/00207713-30-1-225

21. Lee KB, Liu CT, Anzai Y, Kim H, Aono T, Oyaizu H. The hierarchical system of the 'Alphaproteobacteria': description of Hyphomo-nadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst Evol Microbiol 2005; 55:1907-1919. PubMed doi:10.1099/ijs.0.63663-0

22. BAuA. Classification of bacteria and archaea in risk groups. TRBA 2005; 466:205.

23. Ashburner M, Ball CA, Blake JA, Botstein D, But-ler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the un-ification of biology. The Gene Ontology Consor-tium. Nat Genet 2000; 25:25-29. PubMed doi:10.1038/75556

24. Sittig M, Hirsch P. Chemotaxonomic investiga-tions of budding and/or hyphal bacteria. Syst Appl Microbiol 1992; 15:209-222.

25. Kang HS, Lee SD. Hirschia maritima sp. nov., iso-lated from seawater. Int J Syst Evol Microbiol 2009; 59:2264-2268. PubMed doi:10.1099/ijs.0.008326-0

26. Batrakov SG, Nikitin DI, Pitryuk IA. Lipid compo-sition of the gram-negative, budding, seawater bacterium Hyphomonas jannaschiana lacking in phospholipids. Biochim Biophys Acta 1996; 1303:39-46. PubMed

27. Batrakov SG, Nikitin DI, Pitryuk IA. A novel gly-colipid, 1,2-diacyl-3-α-D-glucuronopyranosyl-sn-glycerol taurineamide, from the budding seawater bacterium Hyphomonas jannaschiana. Biochim Biophys Acta 1996; 1302:167-176. PubMed

28. JGI website. http://www.jgi.doe.gov

29. The Phred/Phrap/Consed software package. http://www.phrap.com

30. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821-829. PubMed doi:10.1101/gr.074492.107

31. Han C, Chain P. Finishing repeat regions auto-matically with Dupfinisher. In: Proceeding of the 2006 international conference on bioinformatics & computational biology. Arabnia HR, Valafar H (eds), CSREA Press. June 26-29, 2006: 141-146.

32. Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for us-ing ultra short reads in microbial genome assem-bly and finishing. AGBT, Marco Island, FL, 2008

33. Hyatt D, Chen GL, Locascio PF, Land ML, Lari-mer FW, Hauser LJ. Prodigal Prokaryotic Dynam-ic Programming Genefinding Algorithm. BMC Bioinformatics 2010; 11:119. PubMed doi:10.1186/1471-2105-11-119

34. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for mi-crobial genomes. Nat Methods 2010; 7:455-457. PubMed doi:10.1038/nmeth.1457

35. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in ge-nomic sequence. Nucleic Acids Res 1997; 25:955-964. PubMed doi:10.1093/nar/25.5.955

36. Lagesen K, Hallin PF, Rødland E, Stærfeldt HH, Rognes T, Ussery DW. RNammer: consistent an-notation of rRNA genes in genomic sequences.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=21037002&dopt=Abstract�http://dx.doi.org/10.1128/JB.01205-10�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=18464787&dopt=Abstract�http://dx.doi.org/10.1038/nbt1360�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2112744&dopt=Abstract�http://dx.doi.org/10.1073/pnas.87.12.4576�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16403855&dopt=Abstract�http://dx.doi.org/10.1099/ijs.0.64188-0�http://dx.doi.org/10.1099/00207713-30-1-225�http://dx.doi.org/10.1099/00207713-30-1-225�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16166687&dopt=Abstract�http://dx.doi.org/10.1099/ijs.0.63663-0�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10802651&dopt=Abstract�http://dx.doi.org/10.1038/75556�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19620361&dopt=Abstract�http://dx.doi.org/10.1099/ijs.0.008326-0�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8816851&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8695667&dopt=Abstract�http://www.jgi.doe.gov/�http://www.phrap.com/�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=18349386&dopt=Abstract�http://dx.doi.org/10.1101/gr.074492.107�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20211023&dopt=Abstract�http://dx.doi.org/10.1186/1471-2105-11-119�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20436475&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20436475&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20436475&dopt=Abstract�http://dx.doi.org/10.1038/nmeth.1457�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9023104&dopt=Abstract�http://dx.doi.org/10.1093/nar/25.5.955�

Chertkov et al.

http://standardsingenomics.org 297

Nucleic Acids Res 2007; 35:3100-3108. PubMed doi:10.1093/nar/gkm160

37. Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR. Rfam: an RNA family database. Nucleic Acids Res 2003; 31:439-441. PubMed doi:10.1093/nar/gkg006

38. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol 2001; 305:567-580. PubMed doi:10.1006/jmbi.2000.4315

39. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783-795. PubMed doi:10.1016/j.jmb.2004.05.028

40. Nierman WC, Feldblyum TV, Laub MT, Paulsen IT, Nelson KE, Eisen JA, Heidelberg JF, Alley MR, Ohta N, Maddock JR, et al. Complete genome se-quence of Caulobacter crescentus. Proc Natl Acad Sci USA 2001; 98:4136-4141. PubMed doi:10.1073/pnas.061029298

41. Badger JH, Hoover TR, Brun YV, Weiner RM, Laub MT, Alexandre G, Mrazek J, Ren Q, Paulsen IT, Nelson KE, et al. Comparative genomic evi-dence for a close relationship between the di-morphic prosthecate bacteria Hyphomonas nep-tunium and Caulobacter crescentus. J Bacteriol 2006; 188:6841-6850. PubMed doi:10.1128/JB.00111-06

42. Brilli M, Fondi M, Fani R, Mengoni A, Ferri L, Bazzicalupo M, Biondi EG. The diversity and evo-lution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis. BMC Syst Biol 2010; 4:52. PubMed doi:10.1186/1752-0509-4-52

43. Brown PJ, Hardy GG, Trimble MG, Brun YV. Complex regulatory pathways coordinate cell-cycle progression and development in Caulobac-ter crescentus. Adv Microb Physiol 2008; 54:1-

101. PubMed doi:10.1016/S0065-2911(08)00001-5

44. Merker RI, Smit J. Characterization of the adhe-sive holdfast of marine and freshwater Caulobac-ters. Appl Environ Microbiol 1988; 54:2078-2085. PubMed

45. Quintero EJ, Langille SE, Weiner RM. The polar polysaccharide capsule of Hyphomonas adhae-rens MHS-3 has a strong affinity for gold. J Ind Microbiol Biotechnol 2001; 27:1-4. PubMed doi:10.1038/sj.jim.7000143

46. Smith CS, Hinz A, Bodenmiller D, Larson DE, Brun YV. Identification of genes required for syn-thesis of the adhesive holdfast in Caulobacter crescentus. J Bacteriol 2003; 185:1432-1442. PubMed doi:10.1128/JB.185.4.1432-1442.2003

47. Toh E, Kurtz HD, Jr., Brun YV. Characterization of the Caulobacter crescentus holdfast polysaccha-ride biosynthesis pathway reveals significant re-dundancy in the initiating glycosyltransferase and polymerase steps. J Bacteriol 2008; 190:7219-7231. PubMed doi:10.1128/JB.01003-08

48. Hardy GGR, Allen C, Toh E, Long M, Brown PJ, Cole-Tobian JL. Brun YV. A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole. Mol Microbiol 2010; 76:409-427. PubMed doi:10.1111/j.1365-2958.2010.07106.x

49. Langille SE, Weiner RM. Spatial and temporal deposition of Hyphomonas strain VP-6 capsules involved in biofilm formation. Appl Environ Mi-crobiol 1998; 64:2906-2913. PubMed

50. Larson RJ, Pate JL. Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. J Bac-teriol 1976; 126:282-293. PubMed

51. Wagner JK, Setayeshgar S, Sharon LA, Reilly JP, Brun YV. A nutrient uptake role for bacterial cell envelope extensions. Proc Natl Acad Sci USA 2006; 103:11772-11777. PubMed doi:10.1073/pnas.0602047103

http://standardsingenomics.org/�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17452365&dopt=Abstract�http://dx.doi.org/10.1093/nar/gkm160�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12520045&dopt=Abstract�http://dx.doi.org/10.1093/nar/gkg006�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11152613&dopt=Abstract�http://dx.doi.org/10.1006/jmbi.2000.4315�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15223320&dopt=Abstract�http://dx.doi.org/10.1016/j.jmb.2004.05.028�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11259647&dopt=Abstract�http://dx.doi.org/10.1073/pnas.061029298�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16980487&dopt=Abstract�http://dx.doi.org/10.1128/JB.00111-06�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20426835&dopt=Abstract�http://dx.doi.org/10.1186/1752-0509-4-52�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=18929067&dopt=Abstract�http://dx.doi.org/10.1016/S0065-2911(08)00001-5�http://dx.doi.org/10.1016/S0065-2911(08)00001-5�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16347718&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16347718&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16347718&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11598803&dopt=Abstract�http://dx.doi.org/10.1038/sj.jim.7000143�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12562815&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12562815&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12562815&dopt=Abstract�http://dx.doi.org/10.1128/JB.185.4.1432-1442.2003�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=18757530&dopt=Abstract�http://dx.doi.org/10.1128/JB.01003-08�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20233308&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20233308&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=20233308&dopt=Abstract�http://dx.doi.org/10.1111/j.1365-2958.2010.07106.x�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9687449&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=4425&dopt=Abstract�http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16861302&dopt=Abstract�http://dx.doi.org/10.1073/pnas.0602047103�